Perinuclear Localization and Insulin Responsiveness of GLUT4 Requires Cytoskeletal Integrity in 3T3-L1 Adipocytes

Adilson Guilherme,Masahiro Emoto,Joanne M Buxton,Sahana Bose,Rosanna Sabini,William E Theurkauf,John Leszyk,Michael P Czech

doi:10.1074/jbc.m003432200

Abstract

The GLUT4 glucose transporter resides mostly in perinuclear membranes in unstimulated 3T3-L1 adipocytes and is acutely translocated to the cell surface in response to insulin. Using a novel method to purify intracellular GLUT4-enriched membranes, we identified by mass spectrometry the intermediate filament protein vimentin and the microtubule protein alpha-tubulin as components of these membranes. Immunoelectron microscopy of the GLUT4-containing membranes also revealed their association with these cytoskeletal proteins. Disruption of intermediate filaments and microtubules in 3T3-L1 adipocytes by microinjection of a vimentin-derived peptide of the helix initiation 1A domain caused marked dispersion of perinuclear GLUT4 to peripheral regions of the cells. Inhibition of the microtubule-based motor dynein by brief cytoplasmic acidification of cultured adipocytes also dispersed perinuclear GLUT4 and inhibited insulin-stimulated GLUT4 translocation to the cell surface. Insulin sensitivity was restored as GLUT4 was again concentrated near the nucleus upon recovery of cells in physiological buffer. These data suggest that GLUT4 trafficking to perinuclear membranes of cultured adipocytes is directed by dynein and is required for optimal GLUT4 regulation by insulin.

Highlights

Insulin regulates the cellular uptake of glucose in adipose tissue and muscle by acutely controlling the number of glucose transport proteins present in the cell surface membrane [1,2,3,4]
These data suggest that GLUT4 trafficking to perinuclear membranes of cultured adipocytes is directed by dynein and is required for optimal GLUT4 regulation by insulin
A unifying hypothesis has been derived from the work of many laboratories on these proteins, which suggests that fusion of GLUT4-containing membranes with the plasma membrane involves VAMP2/ syntaxin 4 interaction regulated by SNAP23, MUNC 18, syntaxin 4-interacting protein-1, and other proteins [4]

Summary

Introduction

Insulin regulates the cellular uptake of glucose in adipose tissue and muscle by acutely controlling the number of glucose transport proteins present in the cell surface membrane [1,2,3,4]. Insulin treatment of rat adipocytes prior to disruption of the cells and preparation of these membranes causes a marked decrease in the yield of GLUT4 present in the latter fractions (Fig. 1, A and upper panel of B).

Results

Conclusion